The expression “angiogenic factor” refers to a growth factor that not only stimulates neovascularization and angiogenesis (initiated along with the activation of parent blood vessel endothelial cells) in vivo, but is also mitogenic for endothelial cells in vitro. Examples of angiogenic factors include HGF, VEGF, FGF, and HIF. The first therapeutic application of angiogenic factors was reported by Folkman et al. (N. Engl. J. Med. 285, 1182-1186 (1971)). In later studies, the use of recombinant angiogenic factors such as the FGF family (Science 257, 1401-1403 (1992); Nature 362, 844-846 (1993)) and VEFGF was confirmed as promoting and/or accelerating development of the collateral circulatory tract in animal models of myocardial and hind limb ischemia (Circulation 90, II-228-II-234 (1994)). Furthermore, the present inventors discovered that HGF, like VEGF, functions as an endothelium-specific growth factor (J. Hypertens. 14, 1067-1072 (1996)).
HGF is a cytokine discovered to be a powerful growth-promoting factor for mature stem cells, and its gene has been cloned (Biochem. Biophys. Res. Commun. 122: 1450 (1984); Proc. Natl. Acad. Sci. USA. 83: 6489 (1986); FEBS Letters 22: 231 (1987); Nature 342: 440-443 (1989); Proc. Natl. Acad. Sci. USA. 87: 3200 (1991)). HGF is a plasminogen-related and mesenchymer-derived pleiotropic growth factor, and is known to regulate cell growth and cell motility in various types of cells (Nature 342: 440-443 (1989); Biochem. Biophys. Res. Commun. 239: 639-644 (1997); J. Biochem. Tokyo 119: 591-600 (1996)). It is also an important factor in regulating blastogenesis and morphogenic processes during the regeneration of several organs. For example, HGF is a strong mitogen for epidermal cells such as hepatocytes and keratinocytes (Exp. Cell Res. 196:114-120 (1991)). HGF stimulates angiogenesis, induces cell dissociation, and initiates endothelial cell movement (Proc. Natl. Acad. Sci. USA. 90: 1937-1941 (1993); Gene Therapy 7: 417-427 (2000)). Later studies revealed that HGF not only functions in vivo as a hepatic regeneration factor in the repair and regeneration of the damaged liver, but also has an angiogenic effect and plays an important role in the therapy for or prevention of ischemic and arterial diseases (Symp. Soc. Exp. Biol., 47 cell behavior 227-234 (1993); Proc. Natl. Acad. Sci. USA. 90: 1937-1941 (1993); Circulation 97: 381-390 (1998)). There are reports that administration of HGF to rabbit hind limb ischemia models showed remarkable angiogenesis, improved blood flow, suppression of decrease in blood pressure, and improvement of ischemic symptoms. As a result of these reports, HGF is now considered to be expressed as an angiogenic factor and to function as such.
As its name indicates, HGF was discovered in the liver. However, it actually exists throughout the entire body and has a cell-proliferating action. The vigorous cell division that occurs around an injury to repair the wound is also due to the action of HGF. The dermatology team at Juntendo University discovered that HGF is also a hair growth factor. HGF promotes hair growth by promoting division of hair matrix cells. Administration of HGF to hair matrix cells on scalps which show progressed androgen-related hair thinning is likely to regenerate thick hair.
Furthermore, HGF-induced angiogenesis in rat hearts with non-infarcted and infarcted myocardium (Proc. Natl. Acad. Sci. USA 90: 8474-8478 (1993)), and in rat corneas (Proc. Natl. Acad. Sci. USA 90: 1937-1941 (1993)) has been found in vivo.
Thus, HGF has a multitude of functions, not least of which is its function as an angiogenic factor. Various attempts have been made to utilize HGF in pharmaceutical agents, however, HGF's half-life in the blood has made this a problem. HGF's short half-life of about ten minutes makes maintenance of its blood concentration difficult. Thus, translocation of an effective HGF dose to an affected area is problematic.
VEGF is a dimeric glycoprotein that is mitogenic for endothelial cells and can enhance vascular permeability. VEGF's mitogenic effect is direct and specific to endothelial cells (Biochem. Biophys. Res. Commun., 161, 851-858 (1989)).
HIF promotes the production of erythrocytes and stimulates angiogenesis and erythropoietin (which increases oxygen supplied to the entire body). HIF is also the main transcription factor in the transcriptional activation of VEGF (which increases local oxygen supply), VEGF's receptor, and the genes for various enzymes involved in the glycolytic pathway (which provides resistance to cells by synthesizing ATP in anoxic conditions). HIF-1 is a heterodimer comprising HIF-1α and HIF-1β. HIF-1β (also called Arnt) also forms a heterodimer with the Ah receptor (which is associated with the metabolism of foreign substances such as dioxin) to function in the transcriptional regulation of drug-metabolizing enzyme genes.
In general, gene therapy can be used to treat various recovered clinical diseases (Science 256: 808-813 (1992); Anal. Biochem. 162: 156-159 (1987)) Selection of an appropriate vector for gene transfer is particularly important for successful gene therapy. Viruses, adenoviruses in particular, have been the preferred vectors for gene transfer. However, viral vectors are potentially dangerous when viral infection-associated toxicity, lowered immunity, and mutagenic or carcinogenic effects are considered. An example of an alternative method for gene transfer is the HVJ-liposome-mediated method, which has been reported to be effective in vivo. This method uses liposomes in combination with a viral envelope, and shows hardly any toxicity (Science 243: 375-378 (1989); Anal. NY Acad. Sci. 772: 126-139 (1995)). It has been successfully used for in vivo gene transfer into tissues including the liver, kidney, vascular wall, heart, and brain (Gene Therapy 7: 417-427 (2000); Science 243: 375-378 (1989); Biochem. Biophys. Res. Commun. 186: 129-134 (1992); Proc. Natl. Acad. Sci. USA. 90: 8474-8478 (1993); Am. J. Physiol. 271 (Regulatory Integrative Comp. Physiol. 40): R1212-R1220 (1996)).
Wound healing comprises a succession of events including inflammation, angiogenesis, matrix synthesis, and collagen deposition, leading to re-endothelization, angiogenesis, and formation of granulation tissues (Clark RAF, “Overview and General Consideration of Wound Repair. The Molecular and Cellular Biology of Wound Repair.” Plenum Press. New York (1996) 3-50; Annu. Rev. Med. 46: 467-481 (1995); J. Pathol. 178: 5-10 (1996)). These healing processes are regulated by a number of mitogens and chemotactic factors, including growth factors such as fibroblast growth factor (FGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). However, few studies have focused on the effect of HGF on wound healing (Gastroenterology 113: 1858-1872 (1997)).
Although there are several reports on the transfer of genes such as IGF, PDGF, and EGF into wounds (<Gene Therapy 6: 1015-1020 (1999); Lab. Invest. 80: 151-158 (2000); J. Invest. Dermatol. 112: 297-302 (1999); Proc. Natl. Acad. Sci. USA 91: 12188-12192 (1994)), none of these reports focus on the quantitative and qualitative changes in the number of factors involved in wound healing, or on histopathological effects after HGF gene transfer.
Re-epithilization of a wound occurs by translocation of keratinocytes from the edges of the wound toward its center. In vitro, HGF enhances proliferation, cell growth, and DNA synthesis in keratinocytes cultured under physiological Ca2+ conditions (Exp. Cell Res. 196: 114-120 (1991)). Furthermore, due to enhanced cell turnover, HGF has been found to promote epithelial wound resealing in T84 intestinal monolayers (J. Clin. Invest. 93: 2056-2065 (1994)). In vivo administration of recombinant HGF has been found to promote regeneration of epithelial cells in rat kidneys damaged by anti-tumor agents (Gene Therapy 7: 417-427 (2000)). However, in gastric ulcers produced in rats by cryoinjury, subcutaneous administration of recombinant HGF had no effect on the ulcer-healing rate, despite the increase of human HGF concentration in the serum. Epithelial cell proliferation increased in the borders of the ulcers eight to 15 days after cryoinjury (Gastroenterology 113: 1858-1872 (1997)).
Transient upregulation of TGF-β expression is an important event in wound healing. TGF-β stimulates fibroblasts to produce matrix proteins, matrix protease inhibitors and integrin receptors, thereby modulating matrix formation and intercellular interactions at the wound site (Rokerts A B, Aporn M B: “Transforming growth factor-β. The Molecular and Cellular Biology of Wound Repair” Second Edition, by Clark RAF (Plenum Press. New York, 1996, 275-308)). Abnormal regulation and sustained overexpression of TGF-β1 would presumably contribute to an enhancement of tissue fibrosis, because increased expression of TGF-β1 mRNA has been reported in tissues of patients with cutaneous fibrosis (for example, hypertrophic scars and keloids) (Am. J. Pathol. 152: 485-493 (1998)). Furthermore, TGF-0 neutralizing antibodies not only reduced the cells in the wound granulation tissue of an adult wound, but also improved the architecture of the neodermis (Lancet 339: 213-214 (1992)).
Proteinaceous formulations are generally administered intravenously. HGF has been administered in ischemic disease models both intravenously and intra-arterially (Circulation 97: 381-390 (1998)). Such intravenous or intra-arterial administrations of HGF to animal models have revealed HGF's effectiveness on ischemic or arterial diseases. However, as yet, no conclusion has been reached with regard to a specific and effective method for administration, effective dose, and such. This is particularly so in the case of the HGF protein, due to the above-mentioned problems with half-life and transfer to the affected area. Thus, to date there has been no conclusion regarding an effective method of administration, effective dose, etc.